Anionic amphiphile-independent activation of the phagocyte NADPH oxidase in a cell-free system by p47phox and p67phox, both in C terminally truncated forms. Implication for regulatory Src homology 3 domain-mediated interactions.
Anionic amphiphiles, such as arachidonate, activate the superoxide-producing phagocyte NADPH oxidase in a cell-free system with human neutrophil membrane, which contains cytochrome b558 comprising gp91(phox) and p22(phox), and three cytosolic proteins: p47(phox) and p67(phox), each harboring two SH3 domains, and the small GTPase Rac. Here we show that, even without the amphiphiles, the oxidase is activated in vitro by a C terminally truncated p47(phox), retaining the N-terminal and the two SH3 domains, and the N terminus of p67(phox). When either truncated p47(phox) or p67(phox) is replaced by the respective full-length one, the activation absolutely requires the amphiphiles. The results indicate that both p47(phox) and p67(phox) are the primary targets of the amphiphiles, and that their C-terminal regions play negative regulatory roles. We also find that the truncated p47(phox), but not the full-length one, can bind to p22(phox), a binding required for the oxidase activation. The N-terminal SH3 domain of p47(phox) is responsible for the binding not only to p22(phox), but also to the p47(phox) C terminus. Thus the SH3 domain is accessible in the active p47(phox), but is normally masked in the full-length one probably via intramolecularly interacting with the C terminus. The present findings support our previous proposal of regulatory SH3 domain-mediated interactions.
Hata
K
K
Department of Biochemistry, Kyushu University School of Medicine, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-82, Japan.
Ito
T
T
Takeshige
K
K
Sumimoto
H
H
D002462:Cell Membrane / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils; D010750:Phosphoproteins / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism; D013501:Surface-Active Agents / Q000494:pharmacology*; D018909:src Homology Domains
D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D013501:Surface-Active Agents; C079789:neutrophil cytosol factor 67K; D013481:Superoxides; D012967:Sodium Dodecyl Sulfate; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1